LEAK-respiration

Description

EAK-respiration or LEAK oxygen flux, L, compensating for proton leak, proton slip, cation cycling and electron leak, is a dissipative component of respiration which is not available for performing biochemical work and thus related to heat production. LEAK-respiration is measured in the LEAK state, in the presence of reducing substrate(s), but absence of ADP - abbreviated as L(n) (theoretically, absence of inorganic phosphate presents an alternative), or after enzymatic inhibition of the phosphorylation system, which can be reached with the use of oligomycin - abbreviated as L(Omy). The LEAK state is the non-phosphorylating resting state of intrinsic uncoupled or dyscoupled respiration when oxygen flux is maintained mainly to compensate for the proton leak at a high chemiosmotic potential, when ATP synthase is not active. In this non-phosphorylating resting state, the electrochemical proton gradient is increased to a maximum, exerting feedback control by depressing oxygen flux to a level determined mainly by the proton leak and the H+/O2 ratio. In this state of maximum protonmotive force, LEAK-respiration, L, is higher than the LEAK component of OXPHOS capacity, P. The conditions for measurement and expression of respiration vary (oxygen flux in the LEAK state, JO2L, or oxygen flow, IO2L). If these conditions are defined and remain consistent within a given context, then the simple symbol L for respiratory rate can be used as a substitute for the more explicit expression for respiratory activity.
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LEAK-respiration: concept-linked terminology of respiratory states

LEAK-respiration corresponds to resting, non-phosphorylating electron transfer with a shortcircuit of the proton cycle across the inner mt-membrane due to intrinsic uncoupling or dyscoupling. 2[H] indicates the reduced hydrogen equivalents of CHO substrates and electron transfer to oxygen. H+out are protons pumped out of the matrix phase. Proton leaks (a property of the inner mt-membrane) dissipate energy of translocated protons, and proton slip prevents full translocation of protons across the inner mt-membrane (a property of the proton pumps). Measurement of LEAK-respiration is possible in intact cells by inhibition of the phosphorylation system and in mt-preparations supported by an ET-pathway competent substrate state, exemplifed as the NS-pathway (CI&II-linked substrate supply). Modified after Gnaiger 2014 MitoPathways.

Abstract: Mitochondrial respiratory states have been defined originally by Chance and Williams (1955) as a sequence (from 1 to 5) of titrations and transitions in a respiratory protocol, including State 4 as a LEAK state of respiration obtained after exhaustion of the added ADP. The second state (State 2) is induced by addition of 'high ADP'. Confusion persists in the current literature as to the meaning of State 2, which can be resolved by a transition from a specific protocol-linked to a generalized concept-linked terminology.

The LEAK state

LEAK states of respiration are frequently called State 4, State 4o, basal state, or inactive state. Importantly, State 2 is not a LEAK state according to the classical definition.

Respiration compensating for the proton leak is the main component of LEAK-respiration. If proton leak were the only component involved, it would make sense to simply write leak respiration for the compensatory oxygen flux. Proton slip and cation cycling, however, are also involved to a larger or smaller extent to stimulate LEAK-respiration. The upper case 'LEAK', therefore, should make us aware that this is more accurately considered as an acronym, and cannot be taken as a definitive delineation of the stimulatory mechanism in the LEAK state of respiration.

Note: The distinction between LEAK and ET-capacity helps to clarify the important difference between physiologically uncoupled or pathologically dyscoupled respiration (LEAK) in contrast to noncoupled respiration (ET-capacity).

Protocols for measurement of LEAK-respiration

The LEAK state can be induced experimentally in various ways, which may yield idential estimates of LEAK-respiration, or may show deviations that help to critically assess the proper protocol to be applied in specific cases:

a) LEAK state with ATP

LEAK-respiration in the presence of ATP, L(T), and absence of ATPase activity.

In contradiction to the original definition of State 2 (ROX), yet with reference to Chance and Williams (1956), 'State 2' has later been used for describing this functionally different LEAK state:

‘State 2: substrate added, respiration low due to lack of ADP. .. the controlled respiration prior to addition of ADP, which is strictly termed “state 2”, is functionally the same as state 4, and the latter term is usually used for both states’ (Nicholls & Ferguson 1992).

Thus State 2 was re-defined as functionally the same as State 4. State 2 (Chance and Williams 1955, 1956), however, is substrate-limited residual oxygen consumption at high ADP (ROXD), whereas L(n) and L(T) (State 4) are LEAK-respiration in the absence of adenylates, L(n) (no ADP, no ATP) or presence of ATP, L(T).

To overcome the termonological confusion persisting in the scientific literature, the respiratory coupling states of LEAK-respiration, OXPHOS-capacity and ET-capacity are distinguished from residual oxygen consumption (ROX; Gnaiger 2009).

b) LEAK state with oligomycin

LEAK-respiration induced by inhibition of ATP synthase by oligomycin, L(Omy).

Sequential addition of (1) mitochondria, (2) ADP, and (3) reduced substrates is the basis of the original State 1-2-3 definitions of respiratory states (Chance and Williams 1955 part III, 1956), where State 2 is zero respiration or residual oxygen consumption in the absence of substrate. An alternative protocol is well established, as shown, e.g., by the classical Fig. 5A (Chance and Williams 1955 part I): 600 µM ADP is added after a state described as ‘Aerobic mitochondria plus succinate’. That state was never defined as ‘State 2’ by Brit Chance. Later Estabrook (1967) made this protocol more popular, with addition of substrate before any ADP or ATP was added.

In this alternative protocol, a respiratory LEAK state is induced in isolated mitochondria, permeabilized tissues, or permeabilized cells, adding the mitochondrial preparation to respiration medium containing inorganic phosphate (State 1), then adding reduced substrate (no external adenylates). This second state (Estabrook 1967) is a non-phosphorylating LEAK state, with respiration L(n) (n for no adenylates; Gnaiger 2009), when substrate-saturated respiration compensates for the proton leak (mainly) in the absence of ADP.

Related terms in MitoPedia

State 4 versus State 2

Static head

State 4 is frequently referred to as 'static head' of isolated mitochondria. Equivalence requires testing, if at State 4 (in a protocol defined by Chance and Williams 1955) ATPase activity is actually zero, such that respiration at State 4 is not partially stimulated by partial recycling of ATP to ADP. In the latter case, State 4 respiration would be higher than LEAK-respiration and thus higher than respiration at static head.